Neuroprotective Benefits Of Exercise And MitoQ On Memory Function, Mitochondrial Dynamics, Oxidative Stress, Thiab Neuroinflammation in D-Galactose-Induced Aging Rats Part 2

2.6. Kev npaj cov ntaub so ntswg

Tom qab ua tiav 8 lub lis piam ntawm TE thiab kev ntsuam xyuas tus cwj pwm nco, tag nrho cov nas tau euthanized los ntawm CO2 inhalation, thiab cov ntaub so ntswg hlwb raug sau los ntawm 7 tsiaj hauv txhua pab pawg kho mob.

Kev ntsuam xyuas tus cwj pwm nco yog ib qho cuab yeej zoo rau kev ntsuas kev nco. Nrog rau kev sib raug zoo thiab kev ua haujlwm siab, tib neeg lub cim xeeb tau dhau los ua qhov tseem ceeb. Yog li ntawd, kev sim ntsuas tus cwj pwm nco tuaj yeem pab peb nkag siab txog peb lub cim xeeb thiab ua kom muaj txiaj ntsig zoo los txhim kho thiab tswj peb lub cim xeeb raws li qhov ntsuas tau.

Tshwj xeeb, kev ntsuam xyuas tus cwj pwm nco tuaj yeem ntsuas tib neeg lub peev xwm tau txais, txheej txheem, khaws cia, thiab khaws cov ntaub ntawv. Los ntawm kev sim ntau hom ntaub ntawv xws li hom lus, lej, duab, thiab lwm yam, tib neeg lub cim xeeb tuaj yeem kuaj tau tag nrho. Cov txiaj ntsig kev xeem yuav muab ntau yam qhab nia los pab peb nkag siab txog peb qhov tsis muaj zog thiab lub zog, thiab yuav ua li cas ntxiv dag zog rau peb lub cim xeeb.

Hauv kev xyaum, ntau qhov kev ntsuam xyuas tus cwj pwm nco kuj tseem muab cov lus qhia thiab cov lus qhia. Piv txwv li, kev pw tsaug zog zoo, noj zaub mov kom zoo, kev tawm dag zog kom zoo, thiab kev tawm dag zog lub hlwb tsis tu ncua yog txhua yam muaj txiaj ntsig zoo rau kev txhim kho tib neeg kev nco. Los ntawm cov txheej txheem no, peb tuaj yeem txhim kho peb lub sijhawm khaws cia lub cim xeeb, lub peev xwm rov ua dua lub cim xeeb, thiab lwm yam, kom hloov tau zoo rau lub neej niaj hnub lossis kev xav tau ua haujlwm.

Nyob rau hauv luv luv, kev ntsuam xyuas tus cwj pwm nco muaj txiaj ntsig zoo rau kev nkag siab, ntsuas, thiab txhim kho ib tus neeg lub cim xeeb. Peb kuj yuav tsum tau txais txoj hauv kev tsim nyog los ntxiv dag zog rau peb lub cim xeeb raws li peb qhov xwm txheej tiag tiag, los tsim lub hauv paus ruaj khov rau lub neej ua kom tiav thiab nplua nuj. Nws tuaj yeem pom tias peb yuav tsum txhim kho kev nco, thiab Cistanche tuaj yeem txhim kho kev nco zoo vim Cistanche yog cov khoom siv tshuaj suav tshuaj suav nrog ntau yam tshwj xeeb, ib qho ntawm kev txhim kho kev nco. Cov nyhuv ntawm Cistanche los ntawm ntau yam khoom xyaw uas nws muaj, suav nrog tannic acid, polysaccharides, flavonoid glycosides, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv ntau txoj hauv kev.

Nyem Paub Short-term Nco yuav ua li cas txhim kho

Tom qab sib cais lub paj hlwb cortex thiab lub hippocampus, cov ntaub so ntswg ntawm lub paj hlwb tau flash-kua hauv cov kua nitrogen thiab khaws cia ntawm −80 ◦C kom txog thaum kev soj ntsuam. Lwm 5 tus tsiaj tau siv rau kev soj ntsuam immunohistochemistry (IHC).

Tom qab qhib lub thoracic kab noj hniav, 50 mM phosphate-buffered saline (PBS) tau dhau los ntawm sab laug ventricle rau 10 min, thiab tom qab ntawd tus tsiaj tau perfused nrog 4% paraformaldehyde (PFA) hauv 100 mM PBS.

Tom qab kev txhim kho lub hlwb, lub paj hlwb raug sau tseg, muab tso rau hauv 4% PFA, thiab tsau rau 4 h ntawm 4 ◦C.Cov ntaub so ntswg ruaj khov tau muab tso rau hauv 30% sucrose tov rau 2 hnub thiab tom qab ntawd txiav rau hauv 40 µm-thick slices siv lub cryostat ( Leica Microsystems, Nussloch, Lub teb chaws Yelemees).

2.7. Mitochondria Isolation

Txhawm rau cais cov mitochondria, peb siv Mitochondria Extraction Kit (IMGENEX Corporation, San Diego, CA, USA) raws li cov chaw tsim khoom cov lus qhia. Rau txhua 100 mg ntawm hippocampal cov ntaub so ntswg, 1 mL ntawm homogenizing tsis tau ntxiv.

Tom qab homogenization, cov ntaub so ntswg tau centrifuged ntawm 900 × g rau 10 min ntawm 4 ◦C, thiab cov supernatant tau sau thiab centrifuged dua ntawm 15, 000 × g rau 30 min ntawm 4 ◦C.

Noj cov centrifuged cytosolicfraction, cov cytosol tau sib cais, thiab cov pellet seem yog tov kom huv si hauv 1 mLof ncua kev kawm ntawv, ua ntej centrifuging ntawm 15, 000 × g rau 10 min ntawm 4 ◦C.

Lub supernatant raug tshem tawm, thiab ntxiv 1 mL ntawm kev ncua tsis pub dhau ua ntej sib tov kom huv si thiab centrifuging dua ntawm 15, 000 × g rau 10 min ntawm 4 ◦C.

Cov supernatant raug tshem tawm, thiab cov pellet seem tau yaj hauv 1 mL ntawm kev ua tiav mitochondrial lysis tsis rau 30 min ntawm 4 ◦C. Tom qab ntawd cov mitochondrial extract tau centrifuged ntawm 15, 000 × g rau 5 min ntawm 4◦C, thiab cov supernatant (mitochondrial feem) tau sau.

2.8. Western Blotting
Tag nrho cov protein tau los ntawm kev sib cais mitochondrial tau suav nrog siv txoj kev Bradford. Ib qho sib npaug ntawm cov mitochondrial proteins (30 µg ib txoj kab) yog loadedonto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (8% lossis 12%).

Tom qab electrophoresis, cov proteins raug xa mus rau Polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA, USA).

Thaiv tau ua rau 1 teev ntawm chav tsev kub siv 1 × Tris-buffered saline uas muaj Tween-20 (TBS-T) tov nrog 5% BSA, thiab tom qab ntawd cov membrane tau hnov ​​​​mob nrog cov tshuaj tiv thaiv thawj hmo hmo ntuj ntawm 4 ◦C.

Cov tshuaj tiv thaiv hauv qab no tau siv: Mfn1, Mfn2, Opa1, Drp1, Fis1, p22 phox, p47 phox, gp91phox, SOD-2, catalase, thiab -actin (Santa Cruz Biotechnology, Dallas, TX, USA, dilution: 1 : 1000); thiab TNF- (Abcam, Cambridge, UK, dilution: 1: 1000).

Cov ntaub so ntswg tau muab ntxuav nrog cov ntxhua khaub ncaws (PBS nrog 0.1% Tween 20), tom qab ntawd lawv tau incubated nrog horseradish peroxidase (HRP)-conjugated tshis los tiv thaiv luav theem nrab cov tshuaj tiv thaiv forp22 phox, gp91 phox, thiab TNF. - (Invitrogen, Carlsbad, CA, USA, dilution: 1:5000).

HRPconjugated tshis tiv thaiv nas thib ob cov tshuaj tiv thaiv tau siv rau Mfn1, Mfn2, Opa1, Drp1, Fis1, p47 phox, SOD-2, catalase, thiab -actin (Santa Cruz Biotechnology, dilution: 1: 5000).

Protein qhia qib tau kuaj pom siv ECL Western blotting nrhiav kom tau qhov system (Santa Cruz Biotechnology). Qhov ntom ntom ntawm cov kab mob protein tsim tau raug tshuaj xyuas siv ChemiDoc XRS gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

2.9. Immunohistochemistry (IHC)
Rau kev tshuaj xyuas IHC, txoj kev pub dawb-floating tau siv rau cov ntaub so ntswg xaiv hauv txhua pab pawg kho mob. Tom qab ntxuav cov ntaub so ntswg 3 × rau 10 min txhua hauv 0.01 M PBS, cov ntaub so ntswg raug incubated ntawm 80 ◦C rau 60 min nyob rau hauv lub beaker uas muaj 0.01 M sodium citrate, tom qab ntawd thaiv hauv 10% ib txwm luav ntshav (Millipore Sigma, Burlington, MA, USA).

Cov qauv ntawm cov ntaub so ntswg tau tshwm sim nrog thawj cov tshuaj tiv thaiv anti-p22 phox (Santa Cruz Biotechnology, dilution: 1:500) thiab anti-Glial fibrillary acidic protein (GFAP) (Abcam, dilution: 1: 500) rau 12 h ntawm 4 ◦C. Hnub tom qab, cov ntaub so ntswg tau ntxuav 3 × rau 5 min txhua in0.01 M PBS thiab tom qab ntawd hnov ​​​​mob nrog HRP-conjugated tshis anti-nas thib ob antibody (Santa Cruz Biotechnology, dilution: 1: 5000) ntawm chav tsev kub rau 2 h.

Thaum kawg, cov ntaub so ntswg tau muab tso rau hauv chav tsev kub siv cov khoom siv Vectastain-Elite ABC (Vector Laboratories, Burlingame, CA, USA) cov tshuaj, thiab cov txiaj ntsig tau pom los ntawm kev siv DABPeroxidase Substrate Kit (Vector Laboratories).

Txhua daim ntaub so ntswg tau muab tso rau ntawm lub slideusing nruab nrab nruab nrab (Vector Laboratories) thiab tshuaj xyuas siv lub teeb pom kev zoo (Leica Microsystems).

2.10. Kev Tshawb Fawb Txog Kev Tshawb Fawb

Cov ntaub ntawv raug tshuaj xyuas siv SPSS rau Windows Version 18.0 (SPSS Inc., Chicago, IL, USA). Tag nrho cov txiaj ntsig tau qhia raws li qhov nruab nrab ± SEM. Kev txheeb xyuas qhov tseem ceeb tau txiav txim siab siv ib txoj kev ANOVA. Bonferroni post hoc xeem tau siv rau ntau qhov kev sib piv.Differences raug suav hais tias tseem ceeb ntawm p < 0.05.

3. Cov txiaj ntsig

3.1. Cov teebmeem ntawm Treadmill Exercise thiab MitoQ ntawm Kev Tshaj Tawm ntawm Mitochondrial Dynamics-RelatedProteins hauv Hippocampus ntawm D-Gal-Induced Aging Rats

Peb tau txheeb xyuas tias TE thiab MitoQ cuam tshuam rau mitochondrial fusion-txog yam cuam tshuam (Mfn1, Mfn2, Opa1) thiab fission-related factor (Drp1, Fis1) hauv hippocampus ntawm cov laus nas (Daim duab 1).

Kev nthuav qhia theem ntawm mitochondrial fusion-txog yam muaj feem cuam tshuam sib txawv ntawm cov pab pawg kho mob (Mfn1, F4,34=134.750, p < 0.001; Mfn2, F4,34=80. 816, p < 0.001; Opa1, F4, 34=51.456, p=0.001).

Kev nthuav qhia theem ntawm mitochondrial fission-related factor kuj txawv heev ntawm pab pawg kho mob (Drp1, F4,34=53.448, p < 0.001; Fis1,F4,34=116 .313, p <0.001). Cov txiaj ntsig tom qab hoc tau pom nyob rau hauv daim duab 1.

Piv nrog rau pawg Y-CON, kev qhia ntawm mitochondrial fusion-txog yam cuam tshuam txog kev txo qis hauv pawg D-CON thiab pom tias muaj kev nce ntxiv hauv pawg D-TE, D-MI, thiab D-COMBI pawg.

Ntawm qhov tod tes, kev qhia ntawm mitochondrial fission-related factor Drp1 tau nce hauv pawg D-CON piv nrog pawg Y-CON. Thaum mitochondrial fission signals tau txo qis los ntawm kev kho mob ua ke nrog TE, kev kho MitoQ ib leeg tsis ua rau muaj kev hloov pauv loj.

Lwm qhov mitochondrial fission-related factor, Fis1, tau qhia tib yam kev txo qis li Drp1 tab sis kuj tau txo qis los ntawm kev kho MitoQ ib leeg.

Daim duab 1. Cov txiaj ntsig ntawm treadmill ce thiab mitoquinone (MitoQ) ntawm kev qhia ntawm mitochondrial dynamics ntsig txog cov proteins hauv hippocampus ntawm D-galactose (D-gal) ntxias cov nas laus. (A) Cov neeg sawv cev Western blots ntawm mitochondrial fusion thiab fission-related proteins. (B-F) Densitometric tsom xam ntawm Western blot bands normalized rau -Actin. Cov ntaub ntawv qhia nyob rau hauv Western blot yog txhais tau tias los ntawm xya nas hlwb.

-Actin raug soj ntsuam raws li kev tswj hwm sab hauv. Bonferroni post hoc testwas siv tom qab ANOVA. Tus nqi yog txhais tau tias ± SEM.

ib qho qhia txog qhov sib txawv ntawm pawg tswj xyuas cov tub ntxhais hluas (Y-CON) pawg. b Qhia txog qhov sib txawv ntawm pawg D-galactose (D-CON) pawg. c Qhia txog qhov sib txawv ntawm pawg D-galactose ntxiv rau treadmill ce (D-TE).

d Qhia txog qhov sib txawv ntawm qhov sib txawv ntawm D-galactose ntxiv rau MitoQ (D-MI) pawg (p < 0.05). D-COMBI; D-galactose ntxiv rau TE thiab MitoQ pawg.

3.2. Cov teebmeem ntawm Treadmill Exercise thiab MitoQ ntawm Kev Tshaj Tawm ntawm NADPH Oxidase Subunits hauv Cerebral Cortex thiab Hippocampus ntawm D-Gal-Induced Aging Rats

Peb tau pom qhov sib txawv tseem ceeb ntawm pawg kho mob hauv kev tiv thaiv kab mob p22 phox nyob rau hauv lub paj hlwb cortex thiab hippocampus ntawm cov laus nas (Daim duab 2) (CA3,F4,24=18.786, p < 0.{{11 }}01; cortex, F4, 24=12.662, p < 0.001).

Hauv cortex, pawg D-CON tau qhia txog kev tiv thaiv kev tiv thaiv ntawm p22 phox piv nrog pawg Y-CON, uas txo qis hauv D-TE, D-MI, thiab D-COMBI pawg.

Hauv CA3, nce kev tiv thaiv kab mob ntawm p22 phox tau txo qis hauv D-TE thiab D-COMBI pawg piv rau D-CON. Cov protein qhia theem ntawm NOX subunits p22 phox, gp91 phox, thiab p47phox txawv ntawm pawg (p22 phox, F4,{{10}}}.513, p < 0.{{24} }01; gp91 phox,F4,{16}}.282, p < 0.001; p47 phox, F4,34=69.249, p < 0.001).

Cov txiaj ntsig ntawm post hoctest tau pom qhov nce ntxiv hauv NOX subunit theem hauv D-CON piv rau Y-CON. Txawm li cas los xij, NOX subunit theem tau qis dua hauv txhua pawg kev cuam tshuam piv rau pawg D-CON.

Tshwj xeeb, kev qhia ntawm p22 phox thiab gq91 phox tau txo qis hauv D-TE piv rau pawg D-MI. Cov kev tshawb pom no txhais tau hais tias kev tawm dag zog thiab MitoQ ib leeg tiv thaiv kev laus vim muaj oxidative kev nyuaj siab hauv lub hlwb, tab sis lawv cov kev sib xyaw ua ke ua rau tsis muaj kev cuam tshuam ntxiv.

Daim duab 2. Cov teebmeem ntawm treadmill ce thiab MitoQ ntawm kev qhia ntawm NADPH oxidase subunits nyob rau hauv lub paj hlwb cortex thiab hippocampus ntawm D-gal induced aging nas. (A1) Photomicrographs uas qhia NAPDH p22 phox immunoreactivity nyob rau hauv cerebral cortex thiab hippocampus ntawm D-gal-induced agingrats nyob rau hauv txhua pab pawg neeg. Cov duab plaub dub hauv A1 qhia txog feem ntawm cov duab raws li qhia hauv f–i andp–t. (A2, A3) Optical density quantification of NADPH p22 phox staining in the cerebral cortexand hippocampus.

Bonferroni post hoc xeem tom qab ANOVA. Cov nqi tau nthuav tawm raws li txhais tau tias ± SEM.(n=5 tsiaj). (a–e thiab k–o) Scale bar=100 µm, (f–j thiab p–t) Scale bar=50 µm. (B) Tus Neeg Sawv Cev Sab Hnub Tuaj blot duab rau NADPH oxidase subunits hauv hippocampus.

(C-E) Quantitation ntawm NADPH oxidase subunits hauv hippocampus. Cov ntaub ntawv qhia nyob rau hauv Western blot txhais tau hais tias los ntawm xya nas hlwb. -Actin raug soj ntsuam raws li kev tswj hwm sab hauv. Bonferroni post hoc xeem tau siv tom qab ANOVA. Tus nqi yog txhais tau tias ± SEM.

ib qho qhia txog qhov sib txawv ntawm pawg Y-CON. b Denotesstatistical txawv los ntawm pawg D-CON. c Qhia txog qhov sib txawv ntawm pawg D-TE.(p < 0.05).

3.3. Cov nyhuv ntawm Treadmill Exercise thiab MitoQ ntawm GFAP Qhia hauv Cerebral Cortex thiab Hippocampal Dentate Gyrus ntawm D-Gal-Induced Aging Rats

Peb tau pom qhov sib txawv tseem ceeb ntawm pawg kho mob hauv GFAP immunoreactivity hauv cerebral cortex thiab hippocampal dentate gyrus (DG) ntawm cov laus nas (Daim duab 3)(Cortex, F4,24=13.524, p < 0.001 ; DG, F4, 24=30.364).

Cov txiaj ntsig ntawm kev xeem tom qab hoc tau pom tias kev kho D-gal tau nce GFAP immunoreactivity hauv ob qho tib si cortex thiab DG.

Piv nrog rau pawg D-CON, GFAP qib hauv cortex tau txo qis hauv txhua pawg kev cuam tshuam, thiab GFAP qib hauv DG tau txo qis hauv pawg D-TE thiab D-COMBI, thaum pawg D-MI pom qhov sib txawv hauv GFAP immunoreactivity nkaus xwb. nyob rau hauv lub cortexregion.

Yog li, txawm hais tias TE lossis MitoQ kev kho mob txo D-gal-induced neuroinflammationin cortex, tsuas yog TE cuam tshuam ua rau muaj txiaj ntsig zoo hauv DG.

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